Synthetic system for tunable thresholding of protein signals

Abstract

Disclosed herein include methods, compositions, and kits suitable for use in thresholding of protein signals. There are provided, in some embodiments, synthetic protein circuits that respond to inputs only above or below a certain threshold concentration. In some embodiments, the threshold value itself is tunable. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.

What is claimed is: 
     
       1. A method of thresholding protein signals comprising:
 expressing a synthetic protein circuit in a cell, the synthetic protein circuit comprising: 
 a first input polypeptide comprising a first partner domain and a first polypeptide domain; 
 a second input polypeptide comprising a second partner domain and a second polypeptide domain, wherein the first partner domain is capable of binding the second partner domain, wherein the first polypeptide domain and the second polypeptide domain are capable of associating with each other to constitute a first protein capable of being in a first protein active state when the first partner domain binds the second partner domain; and 
 a thresholding polypeptide comprising a third partner domain and a third polypeptide domain, wherein the third partner domain is capable of binding the second partner domain, wherein the first protein is not in the first protein active state when the third partner domain binds the second partner domain, wherein the first protein in the first protein active state is capable of generating a thresholding output. 
 
     
     
       2. The method of  claim 1 , wherein the expressing comprises administering a nucleic acid encoding the synthetic protein circuit, or
 wherein the expressing comprises administering two or more nucleic acids, and wherein the two or more nucleic acids encode the synthetic protein circuit. 
 
     
     
       3. The method of  claim 2 , wherein the nucleic acid comprises at least one regulatory element for expression of the synthetic protein circuit. 
     
     
       4. The method of  claim 2 , wherein the nucleic acid comprises a vector,
 optionally: wherein the vector comprises an adenovirus vector, an adeno-associated virus vector, an Epstein-Barr virus vector, a Herpes virus vector, an attenuated HIV vector, a retroviral vector, a vaccinia virus vector, or any combination thereof, 
 wherein the vector comprises an RNA viral vector, 
 wherein the vector is derived from one or more negative-strand RNA viruses of the order Mononegavirales, and/or 
 the vector is a rabies viral vector. 
 
     
     
       5. The method of  claim 1 , wherein
 (i) the third polypeptide domain and the second polypeptide domain are incapable of associating to form the first protein in the first protein active state, 
 (ii) the third polypeptide domain and the second polypeptide domain are capable of associating with each other to constitute a first protein capable of being in a first protein inactive state when the third partner domain binds the second partner domain, or 
 (iii) the third polypeptide domain and the second polypeptide domain are capable of associating with each other to constitute a first protein capable of being in a first protein dominant negative state when the third partner domain binds the second partner domain. 
 
     
     
       6. The method of  claim 1 , wherein the thresholding output comprises a first enzymatic reaction with a substrate generating a first product. 
     
     
       7. The method of  claim 6 , wherein the substrate comprises a nucleic acid, a protein, a lipid, or any combination thereof. 
     
     
       8. The method of  claim 1 , wherein the third partner domain and the second partner domain are capable of inducing trans-splicing of the thresholding polypeptide and second input polypeptide when the second partner domain binds the third partner domain, thereby generating a conjugate comprising the second polypeptide domain and the third polypeptide domain, optionally wherein the conjugate is not capable of being in the first protein active state. 
     
     
       9. The method of  claim 1 , wherein the first partner domain has a reduced binding affinity for the second partner domain as compared to the binding affinity of the third partner domain for the second partner domain, or
 wherein the first partner domain binds the second partner domain with a first binding affinity, wherein the third partner domain binds the second partner domain with a second binding affinity, wherein the first partner domain is a variant of the third partner domain that is configured to reduce the first binding affinity, wherein the first partner domain comprises one or more mutations as compared to the third partner domain, and wherein the one or more mutations reduce the first binding affinity by at least 10 percent as compared to the second binding affinity. 
 
     
     
       10. The method of  claim 1 , wherein a thresholding output level is related to a number of molecules of the first protein in the first protein active state. 
     
     
       11. The method of  claim 6 , wherein a thresholding output level is related to a number of molecules of the first product. 
     
     
       12. The method of  claim 1 , wherein the thresholding output is generated in response to a first input. 
     
     
       13. The method of  claim 12 , wherein a first output is generated in response to the first input, wherein a first output level positively correlates with a first input level and wherein the thresholding output is generated in response to the first output. 
     
     
       14. The method of  claim 13 , wherein the first output comprises a first protease in a first protease active state and wherein the first output level is related to a number of molecules of the first protease in a first protease active state. 
     
     
       15. The method of  claim 1 , wherein the thresholding output level generated below a threshold first input level is less than about 5% as compared to the thresholding output level generated at or above the threshold first input level. 
     
     
       16. The method of  claim 1 , wherein no thresholding output is generated below a threshold second input polypeptide concentration, or
 wherein the thresholding output level generated below a threshold second input polypeptide concentration is less than about 5% as compared to the thresholding output level generated at or above the threshold second input polypeptide concentration. 
 
     
     
       17. The method of  claim 1 , wherein the synthetic protein circuit is capable of inducing cell death in the presence of the aberrant signaling of a first signal transducer, or when a first level of activation of the first signal transducer is above a first signal transducer activation threshold. 
     
     
       18. A synthetic protein circuit, comprising:
 a first input polypeptide comprising a first partner domain and a first polypeptide domain; 
 a second input polypeptide comprising a second partner domain and a second polypeptide domain, wherein the first partner domain is capable of binding the second partner domain, wherein the first polypeptide domain and the second polypeptide domain are capable of associating with each other to constitute a first protein capable of being in a first protein active state when the first partner domain binds the second partner domain; and 
 a thresholding polypeptide comprising a third partner domain and a third polypeptide domain, wherein the third partner domain is capable of binding the second partner domain, wherein the first protein is not in the first protein active state when the third partner domain binds the second partner domain, wherein the first protein in the first protein active state is capable of generating a thresholding output, 
 wherein the third partner domain and the second partner domain are capable of inducing trans-splicing of the thresholding polypeptide and second input polypeptide when the second partner domain binds the third partner domain, thereby generating a conjugate comprising the second polypeptide domain and the third polypeptide domain, optionally wherein the conjugate is not capable of being in the first protein active state. 
 
     
     
       19. The synthetic protein circuit of  claim 18 , wherein the first partner domain, second partner domain, and/or third partner domain comprise an intein-N, an intein-C, a fragment thereof, or any combination thereof. 
     
     
       20. The synthetic protein circuit of  claim 18 , wherein (1) the first partner domain comprises the amino acid sequence of SEQ ID NO: 9; (2) the second partner domain comprises the amino acid sequence of SEQ ID NO: 8; and/or (3) the third partner domain comprises the amino acid sequence of any one of SEQ ID NOs: 7, 10, and 11.